Optimizing pharmacokinetics and bioavailability of drugs, especially biological agents with limited life-time in the blood, will improve outcome in a number of clinical settings. Drug delivery systems including polyethylene glycol (PEG)-stealth chemistry and liposomal and polymeric carriers are devised to achieve this goal. Among these carriers, red blood cells (RBC) seem very attractive, natural carriers for sustained delivery of drugs acting upon intravascular targets. We devised an original approach for involving biocompatible coupling of therapeutic proteins to the surface of RBC. To avoid ex vivo loading and re-infusion of modified RBC, we fused these cargoes with a single chain fragment of a monoclonal antibody (mAb) to mouse RBC (scFv). These fusions: i) bind to RBC in the bloodstream after injection; ii) safely circulate for a prolonged time; and, iii) get activated loclly by pathological mediators and confer protective effects not seen with non-targeted cargoes in mouse models of human diseases. To enable clinical translation of this approach, we took an advantage of a unique phage-display library created from a macaque previously immunized with human RBC to isolate primate scFv binding to human and pig RBC and fused it with therapeutic moiety, producing functional RBC-targeted fusions that can be tested, optimized and used in these animal species. Capitalizing on these successes, we now propose to employ RBC for carriage of a novel targeted biotherapeutic designed in this study, scFv-fusion with thrombomodulin (TM). Our recent publication and pilot data show that scFv/TM exerts unique anti-thrombotic and anti-inflammatory benefits in mouse models. The goal of this project is to better understand the anti-thrombotic and anti-inflammatory effects of this novel targeted agent, define its mechanisms of action, and to convert it into a primate scFv/TM chimeric protein that binds both pig and human RBC and bring our studies towards clinical application. We will pursue three Aims: Aim 1: Assess scFv/TM benefit/risk ratio. In this Aim we will test the hypothesis that RBC-targeted scFv/TM is more effective and safe than either sTM or APC in mouse models of thrombosis and inflammation. Aim 2: Define mechanisms of action of scFv/TM. We will differentiate thrombin and APC-mediated vs. direct effects of scFv/TM, test the role of the RBC glycocalyx in scFv/TM regulation and define the role of the cytokine-quenching lectin vs. the thrombin-quenching EGF domains of TM in mediating its anti-thrombotic vs. anti-inflammatory activities. Aim 3: Translate scFv/TM into a clinically useful format. We will produce primate scFv/TM fusion, identify the human RBC binding site, test scFv/TM binding and functionality with human and pig RBC, examine its effects on RBC biocompatibility, circulation and effects in pigs.